Modified vaccinia virus ankara for the vaccination of neonates

ABSTRACT

The invention relates inter alia to a method for inducing a long-term protection in an animal against foreign antigens and tumor antigens comprising the step of administering to the animal at least one factor selected from type I interferons and Flt-3, and to a method for inducing a long-term increase of the number of dendritic cells in an animal comprising the step of administering to the animal a factor selected from type I interferon and Flt-3 and to a method of inducing or enhancing the maturation and/or for the activation of the immune system of an animal comprising the step of administering to the animal a factor selected from type I interferon and Flt-3.

The invention concerns the use of a virus for the preparation of amedicament for the vaccination or treatment of a neonatal or prenatalanimal, including a human, wherein the virus is capable of infecting thecells of the neonatal or prenatal animal, including a human, but notcapable of being replicated to infectious progeny virus in the neonatalor prenatal animal, including a human. The virus may be a ModifiedVaccinia Virus Ankara.

In particular, the invention concerns the vaccination of neonatesagainst infections with viruses belonging to the same virus group as thevirus used for vaccination. Moreover, the invention concerns thevaccination of neonates against antigens selected from foreign antigensand tumour antigens, wherein the tumour antigen and/or the foreignantigen are different from the antigens associated with the virus. Theinvention further concerns the use of viruses as defined above toincrease the level of factors which activate dendritic cells or theirprecursor cells and/or to increase the number of dendritic cells ortheir precursor cells and/or to increase the production and/or cellularcontent of an interferon (IFN) or IL-12.

BACKGROUND OF THE INVENTION

The natural environment of animals and human beings contains a largevariety of infectious agents such as viruses, bacteria or fungi. Many ofthese infectious agents may cause diseases in the infected hosts. Undernormal circumstances the infected host recovers from the disease inducedby the infectious agent after a certain period of time. This recovery isdue to the immune system of an animal or a human being.

The immune system is the part of a human or animal body that isresponsible for eliminating the infectious agent. The immune response isdivided into a specific and an unspecific (innate) reaction althoughboth cooperate closely. The unspecific immune response is the immediatedefence against a wide variety of foreign substances and infectiousagents. In the innate immune response against viruses, Interferon(IFN)-α and IFN-β are absolutely essential to control the initial virusreplication and to activate natural killer (NK) cells for immediatekilling of infected cells. Intracellular bacterial or parasiticpathogens induce IL-12 that up regulates IFN-γ in NK cells and/or some Tcell subsets. IFN-γ activated NK cells can now kill intracellularpathogens. Moreover, IFN-γ also activates macrophages and enables themto kill internalized pathogens.

By far the richest source of IFN-α/β, on a per cell basis, are dendriticcells (DC), a specialized cell population strategically distributedthroughout the body. Plasmacytoid DC or CD11c⁺ CD8⁺ DC are among thebest producers of IFN-α/β. CD8⁺ DC that are infected with intracellularnon-viral pathogens are the crucial cells able to secrete IL-12essential for the early steps in immune defense.

A specific immune response can be induced against a particular foreignsubstance (antigen) after a lag phase, when the organism is challengedwith this substance for the first time. The initiation of the specificimmune response is coordinated by DC, too. There is a constant trafficof these cells from the periphery to the secondary lymphoid organs, thelymph nodes or spleen where naïve T and B cells recirculate. Antigenthat is carried by DC to these organs, enables activation of naïve T-and B cells to become effector T- and B cells. For this, DC's not onlycarry the antigen, but the plasticity of pathogen recognition allowsdifferent gene activation in DC and thus a pathogen adjusted priming ofT cells.

The specific immune response is highly efficient and is responsible forthe fact that an individual who recovers from a specific infection isprotected against this specific infection. Thus, a second infection withthe same or a very similar infectious agent causes much milder symptomsor no symptoms at all, since there is already a “pre-existing specificimmunity” to this agent. Such immunity and the immunological memory,respectively, persist for a long time, in some cases even lifelong.Accordingly, the induction of an immunological memory can be used forvaccination, i.e. to protect an individual against infection with aspecific pathogen.

For vaccination the immune system is challenged with a vaccine whichitself is less harmful than the pathogenic agent against which an immuneresponse is to be induced. The vaccine comprises or expresses epitopesthat are found in or expressed by the agent against which thevaccination is done. The organism, thus, is immunized against the agentcontaining the epitope that is part of the vaccine.

Typical vaccines are attenuated or inactivated viruses (e.g. the polioor small poxvirus vaccines), recombinant proteins (e.g. recombinantHepatitis B virus S-protein), heat inactivated bacterial toxins(Clostridium tetani toxin) or polysaccharides of the bacterial capsulewall (Streptococcus pneumoniae).

Since infectious diseases might lead to very critical conditions innewborns and nursing infants, there is an interest to vaccinate childrenor newborn animals as early as possible. Examples for conditions againstwhich a vaccination is desirable are poxvirus infections, includingsmallpox. However, the attempts to successfully vaccinate newborns arehampered by the fact that the immune system of newborn mammals is notyet mature. The immune system of neonatal infants and mammalian animalsis thought to mature gradually over a certain period of time. For humansthe maturation occurs during the first year of life. This is the reasonfor the fact that the neonatal age group is left open to variousinfections during this first year (Gans et al., J. Am. Med. Assoc.(1998) 280, 527-532). More particularly, the neonatal infants haveimpaired B-cell function, deficiencies in primary antigen presentationby dendritic cells and limited T-cell proliferation (Gans et al., J. Am.Med. Assoc. (1998) 280, 527-532). Shortly after birth the levels of Tcells in the spleen are 1,000 fold lower than in adults. In order toachieve at least a weak immunization it was suggested to use eitherreplicating viruses or formulations comprising an adjuvant forimmunization. However, with replication viruses there is always the riskthat the immature immune system may become overwhelmed by virusinfection or live viral vaccines since T cells are necessary for viralclearance (Hassett et al., J. Virol. (1997) 71, 7881-7888). Since thereis a reduced production of cytokines by Th-1 helper T cells in neonates,the response by the infants is predominantly Th-2. Consequently,cytotoxic T cells are not recruited and viral clearance is not achieved.

The situation in mammalian animals is very similar to the situation inhumans, i.e. the immune system after birth is not yet mature. In newbornmice, the number of splenic CD4+ T cells and CD8+ T cells is,respectively, 80,000-fold and 1000-fold lower than in spleens of adults.Moreover, the Interferon (IFN) producing system is immature in thesemice. Therefore, neonatal mice are unable to efficiently control theexpansion of intracellular pathogens by IFN at the site of infection. Inaddition, the low number and possibly inadequate activation stage ofimmune cells are too limited to cope with the rapidly expandingpathogens or replicating viruses used for vaccination.

Due to the risk associated with live viral vaccines, it is notrecommended to vaccinate neonatal animals, including humans, withreplicating viruses. E.g. it is recommended not to vaccinate newbornsagainst smallpox with the vaccinia virus strains that have been useduntil the eradication of smallpox, such as strains Elstree, Copenhagenand NYCBH. According to recent recommendations in the USA, babiesyounger than 12 months of age should not get the smallpox vaccinescommercialized so far.

The vaccination of neonates with formulations comprising an adjuvant hasthe disadvantage that numerous harmful substances are introduced intothe body. Thus, a vaccination in human neonates is only done inemergency cases, e.g. in case of the Hepatitis B virus infection.

In summary, it is to be noted that the immune system is not mature atbirth. Since the vaccination with replication competent viruses, orformulations comprising an adjuvant have significant disadvantages,infants are not vaccinated before the age of 2 months in Germany(Empfehlung der Ständigen lmpfkommission STICO, 2001) or 6 weeks in theUSA (ACIP “Recommended Childhood Immunization Schedule, United States”).

The delay in the development of the immune system is compensated in partby the transfer of maternal antibodies from the mother to the nursinginfant during pregnancy or by breastfeeding. However, not all infantsare breastfed for various reasons. Thus, there is a very critical periodof time of about 6-8 weeks in humans during which the infant having animmature and thus a not fully functional immune system does not receivematernal antibodies and during which a vaccination is usually notsuccessful or too dangerous.

The situation is very similar in mammalian animals, in particular foreconomically important animals such as cows or companion animals such ascats and dogs. To reduce costs, the amount of milk the calf receivesfrom the mother is often drastically reduced. Instead, the calf receivesa mixture of milk powder, starter and specific concentrated feed,sometimes already in the first week after birth. Consequently, the calfdoes not receive the necessary amount and variety of maternalantibodies, so the immature immune system is very susceptible toinfections. Furthermore, farmers who breed calves and those who raisethem for meat production are often not the same. At 4 to 6 weeks of agecalves from different breeder farms are pooled and shipped to otherfarms for meat production. At this time, when maternal antibodies arelow and the immune system is not fully developed, the animals areexposed to new infectious agents under stress conditions. This increasesthe risk for infections that could be prevented by vaccination. Asimilar situation can be found in cat or dog breeding facilities wherethe risk for infection is high.

OBJECT OF THE INVENTION

It is the object of the present invention to provide a means tovaccinate newborn humans and animals, respectively, against foreignantigens and antigens that are associated with diseases in each group,respectively. More particularly, it is the object of the presentinvention to provide a means allowing the accelerated maturation of theimmune system of newborn animals and humans. It is a further object ofthe present invention to provide a means that allows vaccinatingneonatal animals, including humans, against poxvirus infections, inparticular against smallpox.

SUMMARY OF THE INVENTION

What we therefore believe to be comprised by our invention may besummarized inter alia in the following words:

A method for inducing a long-term protection in an animal, including ahuman, against foreign antigens and tumor antigens, comprising the stepof administering to the animal, including a human, at least one factorselected from type I interferons and Flt-3; such a

method wherein the type I Interferon is an Interferon-alpha, or aderivative thereof, or an interferon-beta, or a derivative thereof; sucha

method wherein two or more of the factors are administered to theanimal; such a

method wherein the long-term protection lasts at least 5 days after theadministration of the factor(s); such a

method wherein the foreign antigen is an infectious agent; such a

method wherein the infectious agent is selected from a virus, abacterium, a prion, a parasitic agent, a eukaryotic unicellular ormulticellular infectious agent and a fungus; such a

method wherein the animal is a neonatal or prenatal animal; such a

method wherein the animal is an adult animal; such a

method wherein the animal is immune compromised; such a

method wherein before, after or simultaneous to the administration ofthe factor, a virus is administered to the animal, wherein the virus iscapable of infecting the cells of the animal but not capable of beingreplicated in these cells to infectious progeny virus; such a

method wherein the virus is a DNA virus; such a

method wherein the DNA-virus is selected from DISC-Herpes virus andModified Vaccinia virus Ankara (MVA); such a

method wherein the MVA virus is MVA-BN, deposited at the EuropeanCollection of Animal Cell Cultures (ECACC) with the deposition numberV00083008, and derivatives thereof; such a

method wherein the virus genome comprises at least one heterologousnucleic acid; such a

method wherein the animal is a human; such a

pharmaceutical composition for administration to an animal, including ahuman, which is designed to protect against foreign antigens and/ortumor antigens, comprising either (i) one or more factors, selected fromtype I interferons and Flt-3 and, optionally, a virus which is capableof infecting the cells of an animal, including a human, but not capableof being replicated in such cells to infectious virus progeny, or (ii)two or more factors selected from type I interferon and Flt-3 and,optionally, a virus which is capable of infecting the cells of ananimal, including a human, but not capable of being replicated in suchcells to infectious virus progeny; such a

kit comprising the either (i) one or more factors, selected from type Iinterferons and Flt-3 and a virus which is capable of infecting thecells of an animal, including a human, but not capable of beingreplicated in such cells to infectious virus progeny or (ii) two or morefactors selected from type I interferon and Flt-3 and, optionally, avirus which is capable of infecting the cells of an animal, including ahuman, but not capable of being replicated in such cells to infectiousvirus progeny, such a

method for inducing an increase of the number of dendritic cells in ananimal, including a human, comprising the step of administering to theanimal, including a human, a factor selected from type I interferon andFlt-3; such a

method wherein the dendritic cells (DC) may be conventional DC (cDC) andplasmacytoid pre DC (pDC); such a

method wherein the type I Interferon is Interferon-alpha orinterferon-beta; such a

method wherein two or more of the factors are administered to theanimal; such a

method wherein the number of DC is increased for at least 5 days afterthe administration of the factor(s); such a

method wherein the animal is a neonatal or prenatal animal; such a

method wherein the animal is an adult animal; such a

method wherein the animal is immune compromised; such a

method wherein before, after or simultaneous to the administration ofthe factor, a virus is administered to the animal, wherein the virus iscapable of infecting the cells of the animal but not capable of beingreplicated in these cells to infectious progeny virus; such a

method wherein the virus is a DNA virus; such a

method wherein the DNA-virus is selected from DISC-Herpesvirus andModified Vaccinia virus Ankara (MVA); such a

method wherein the MVA virus is MVA-BN, deposited at the EuropeanCollection of Animal Cell Cultures (ECACC) with the deposition numberV00083008, and derivatives thereof; such a

method wherein the virus genome comprises at least one heterologousnucleic acid; such a

method wherein the animal is a human; such a

method of inducing or enhancing the maturation and/or for the activationof the immune system of an animal, including a human, comprising thestep of administering to the animal, including a human, a factorselected from type I interferon and Flt-3; such a

method wherein the type I Interferon is Interferon-alpha orinterferon-beta; such a

method wherein two or more of the factors are administered to theanimal; such a

method wherein the animal is a neonatal or prenatal animal; such a

method wherein the animal the immune system of which is to be activatedis an adult animal; such a

method wherein the animal is immune compromised; such a

method wherein before, after or simultaneous to the administration ofthe factor, a virus is administered to the animal, wherein the virus iscapable of infecting the cells of the animal but not capable of beingreplicated in these cells to infectious progeny virus; such a

method wherein the virus is a DNA virus; such a

method wherein the DNA-virus is selected from DISC-Herpesvirus andModified Vaccinia virus Ankara (MVA); such a

method wherein the MVA virus is MVA-BN, deposited at the EuropeanCollection of Animal Cell Cultures (ECACC) with the deposition numberV00083008, and derivatives thereof; such a

method wherein the virus genome comprises at least one heterologousnucleic acid; such a

method wherein the animal is a human.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention it was unexpectedly found that it ispossible to safely and efficiently vaccinate and/or treat neonatal orprenatal animals, including humans, with viruses that are capable ofinfecting cells of the neonatal or prenatal animal, including a human,but not capable of being replicated in said cells to infectious progenyvirus. In particular it has been shown that the viruses used accordingto the present invention, such as MVA, in particular MVA-BN and itsderivatives (see below), can be administered to newborns without showingany harmful effects. The vaccination of the animal with the virus leadsto a specific immune response against the virus used for vaccinationand/or to a general vaccination against foreign antigens and tumorantigens as explained below in more detail. Moreover, the viruses usedaccording to the present invention lead to an induction and/orenhancement of the maturation of the immune system, which is associatedwith an increase in the number of dendritic cells and factors such asInterferons. Vaccination with the viruses used according to the presentinvention is possible even if the formulation administered to the animaldoes not comprise an adjuvant.

In summary, the viruses that are used according to the present invention(i) elicit an effective immune response in neonates, (ii) can beadministered without the need of an adjuvant and (iii) do not bear therisk of overwhelming the organism.

According to the present invention the protective effect is exerted forat least 5 days, e.g. for at least 7, 14 or 28 days after the firstvaccination.

Viruses that are “capable of infecting cells” are viruses harboringviral surface structures capable of interacting with the host cells tosuch an extent that the virus or at least the viral genome becomesincorporated into the host cell. Although the viruses used according tothe present invention are capable of infecting the host cell, they arenot capable of being replicated to infectious progeny virus in theinfected cells. In the context of the present invention the term “virusnot capable of being replicated to infectious progeny virus in saidcells” refers to viruses the genome of which is at least partiallytranscribed and translated into viral proteins or even replicated,however, not packaged into infectious viral particles. Thus, the virusesused according to the present invention are viruses leading to abortiveinfections in the host. Abortive infections may occur for two reasons:according to the first alternative a cell may be susceptible toinfection but it may be nonpermissive for multiplication of the virus;e.g. due to the fact that not all necessary viral genes formultiplication of the virus in said cell are expressed and/or present inthe viral genome. An example of this type of virus according to thepresent invention in human cells is Modified Vaccinia Virus Ankara(MVA), which is explained in more detail below. According to the secondalternative an abortive infection may also result from infection ofcells with defective viruses, which lack a full complement of viralgenes. An example of such a virus according to the present invention inhuman cells is DISC-HSV1 (disabled single-cycle Herpes simplex virus),i.e. a Herpes simplex virus, which is restricted to a single cycle ofinfection (Dilloo et al., Blood 1997, 89: 119-127). This virus lacks thegene for the essential glycoprotein H (gH), but can be grown to hightiter in a complementing cell line expressing gH. In noncomplementingcell lines that are permissive for herpes virus growth, it is restrictedto a single cycle of replication, leading to the release ofnoninfectious virus. The term “not capable of being replicated” mayrefer to viruses that do not replicate in the cells of the vaccinatedanimal. However, viruses showing a minor residual replication activitythat is controlled by the immature immune system of the neonate arewithin the scope of the present application.

The virus according to the present invention may be any virus that iscapable of infecting cells of the animal, but not capable of beingreplicated to infectious progeny virus in said cells. It is to beunderstood, that a virus capable of infecting cells of a first animalspecies but not capable of being replicated to infectious progeny virusin said cells may behave differently in a second animal species. Inhumans for example, MVA-BN virus and its derivatives (see below) arecapable of infecting cells, but are not capable of being replicated toinfectious progeny virus in said human cells. The same viruses, however,are very efficiently replicated in chickens, i.e. MVA-BN virus iscapable of infecting chicken cells, and replicating to infectiousprogeny virus in chicken cells. One skilled in the art understands whichvirus to choose for a specific animal species. U.S. Pat. No. 6,761,893discloses a test using murine strain AGR129, that allows determinationof whether a virus is capable, or not, of being replicated in a neonatalor prenatal animal. The results obtained in this murine model areindicative for humans. Thus, the term “not capable of being replicatedto infectious progeny virus” as used in the present applicationcorresponds to the term “failure to replicate in vivo” as used for micein U.S. Pat. No. 6,761,893. More details on this test are given below.The viruses according to the present invention are preferably capable ofbeing replicated in at least one type of cells of at least one animalspecies. Thus, it is possible to amplify the virus prior toadministration to the animal that is to be vaccinated and/or treated. Byway of example reference is made to MVA-BN that can be amplified inchicken embryo fibroblast (CEF) cells, but is not capable of beingreplicated to infectious progeny virus in the neonatal or prenatalhuman. In this context it is to be noted that chemically or physicallyinactivated viruses do not have all the properties of this embodiment.Inactivated viruses are capable of infecting the cells of the neonatalor prenatal animal, including a human and not capable of beingreplicated to infectious progeny virus in the neonatal or prenatalanimal, including a human. However, inactivated viruses are not capableof replicating in at least one type of cells of at least one animalspecies.

The virus may be a DNA virus. For mammalian cells, in particular forhuman cells, the DNA virus may be selected from DISC-Herpesviruses andModified Vaccinia virus Ankara (MVA).

Modified Vaccinia Ankara (MVA) virus is related to Vaccinia virus, amember of the genera Orthopoxvirus in the family of Poxyiridae. MVA hasbeen generated by 516 serial passages of the Ankara strain of vacciniavirus (CVA) (for review see Mayr, A., et al. Infection 3, 6-14 [1975])in CEF. As a consequence of these long-term passages the resulting MVAvirus deleted about 31 kilobases of its genomic sequence and, therefore,was described as highly host cell restricted to avian cells (Meyer, H.et al., J. Gen. Virol. 72, 1031-1038 [1991]). It was shown, in a varietyof animal models that the resulting MVA was significantly avirulent(Mayr, A. & Danner, K. [1978] Dev. Biol. Stand. 41: 225-34).Additionally, this MVA strain has been tested in clinical trials asvaccine to immunize against a human smallpox disease (Mayr et al., Zbl.Bakt. Hyg. I, Abt. Org. B 167, 375-390 [1987], Stickl et al., Dtsch.med. Wschr. 99, 2386-2392 [1974]). These studies involved over 120,000humans, including high risk patients, and proved that, compared toVaccinia based vaccines, MVA had diminished virulence or infectiousnesswhile it maintained good induction of immunity.

Examples of strains according to the present invention are MVA 575,deposited at the European Collection of Animal Cell Cultures (ECACC)with the deposition number V00120707, MVA-572 deposited at ECACC withthe deposition number V94012707 and MVA-BN, deposited at the sameinstitution with the deposition number V000083008, and derivativesthereof, in particular if it is intended to vaccinate/treat humans. Anexample of a stain that may be used in humans is MVA-BN and itsderivatives.

By way of example the properties of MVA strains, in particular theproperties of strains that may be administered to humans, such as MVA-BNand its derivatives, can be summarized as follows:

-   -   (i) capability of reproductive replication in chicken embryo        fibroblasts (CEF) and in Baby Hamster Kidney cells (BHK), but no        capability of reproductive replication in the human cell line        HaCaT,    -   (ii) failure to replicate in vivo,    -   (iii) induction of a higher level of immunity compared to the        known strain MVA 575 (ECACC V00120707) in a lethal challenge        model and/or    -   (iv) induction of at least substantially the same level of        immunity in vaccinia virus prime/vaccinia virus boost regimes        when compared to DNA-prime/vaccinia virus boost regimes.

MVA strains according to the present invention may possess property (ii)above, i.e., failure to replicate in the organism, which is to bevaccinated or treated and/or in the corresponding test system asexplained below, and optionally, one additional of the above properties.The MVA strains may have three of the above properties. An example of anMVA strain having all of the above properties in humans is MVA-BN.Derivatives of MVA-BN may be derivatives having in addition to feature(ii), at least one additional of the above properties, at least twoadditional of the above properties, or all of the above properties.

Reference is made to U.S. Pat. No. 6,761,893 for detailed informationregarding assays used to determine whether a MVA strain has one, ormore, of the above features (i) to (iv). The publication also discloseshow viruses having the desired properties can be obtained. Inparticular, U.S. Pat. No. 6,761,893 provides: a detailed definition ofthe features of MVA-BN and a derivative thereof; a detailed descriptionof biological assays used to determine whether an MVA strain is MVA-BNor a derivative thereof; and methods to obtain MVA-BN or a derivativethereof. In other words, the features of MVA-BN; the description ofbiological assays allowing to evaluate whether a MVA strain is MVA-BN ora derivative thereof; and methods describing how to obtain MVA-BN, or aderivative thereof, are disclosed in U.S. Pat. No. 6,761,893.

The procedures disclosed in U.S. Pat. No. 6,761,893 are summarizedbelow. This summary does not limit the relevance of this disclosure, thefull extent of which is incorporated by reference.

The term “not capable of reproductive replication” in the cell lineHaCaT (Boukamp et al. 1988, J Cell Biol 106(3): 761-71) is used in thepresent application as defined in U.S. Pat. No. 6,761,893. Thus, a virusthat is “not capable of reproductive replication” in the cell line HaCaTis a virus that shows an amplification ratio of less than 1 in the humancell line HaCaT. The amplification rate of the virus used as a vectoraccording to the invention may be 0.8 or less in the human cell lineHaCaT. The “amplification ratio” of a virus is the ratio of virusproduced from an infected cell (Output) to the amount originally used toinfect the cells in the first place (Input) (“amplification ratio”). Aratio of “1” between Output and Input defines an amplification statuswherein the amount of virus produced from the infected cells is the sameas the amount initially used to infect the cells. The term “derivatives”of the viruses as deposited under ECACC V00083008 may refer to virusesshowing essentially the same replication characteristics as thedeposited strain but showing differences in one, or more parts, of itsgenome. Viruses having the same “replication characteristics” as thedeposited virus replicate with similar amplification ratios as thedeposited strain in CEF cells and the cell lines BHK, HeLa, HaCaT and143B. These viruses also show a similar replication in vivo, asdetermined in the AGR129 transgenic mouse model (see below).

The term “failure to replicate in vivo” is used in the presentapplication as defined in U.S. Pat. No. 6,761,893. Thus, the term refersto viruses that do not replicate in humans and in the murine model, asexplained in U.S. Pat. No. 6,761,893. The mice used in U.S. Pat. No.6,761,893 are incapable of producing mature B- and T-cells (AGR 129mice). In particular, MVA-BN and its derivatives, do not kill AGR129mice within mean (average) time periods of at least 45 days, or even ofat least 60 days or even of at least 90 days after the infection of themice with 10⁷ pfu virus administered intraperitonealy. The viruses thatshow “failure to replicate in vivo” are further characterized in that novirus can be recovered from organs or tissues of the AGR129 mice 45 daysor 60 days or even 90 days (mean (average) values) after the infectionof the mice with 10⁷ pfu virus administered intraperitonealy.

Instead of AGR129 mice, another mouse strain may be used which isincapable of producing mature B and T cells and, as such, is severelyimmune compromised and highly susceptible to a replicating virus.

The details of the lethal challenge experiment used to determine whethera MVA strain has “a higher immunogenicity compared to the known strainMVA 575” are explained in U.S. Pat. No. 6,761,893. In such a lethalchallenge model unvaccinated mice die after the infection withreplication competent vaccinia strains such as the Western Reservestrain L929 TK+ or IHD-J. The infection with replication competentvaccinia viruses is referred to as “challenge” in the context ofdescription of the lethal challenge model. Four days after the challengethe mice are usually killed and the viral titer in the ovaries isdetermined by standard plaque assays using VERO cells. The viral titeris determined for unvaccinated mice and for mice vaccinated with MVA-BNand its derivatives. More specifically MVA-BN and its derivatives arecharacterized in that, in this test after the vaccination with 10²TCID₅₀/ml virus the ovary virus titers are reduced by at least 70%, orby at least 80% or by even at least 90% (mean (average) values) comparedto unvaccinated mice.

In a further embodiment the viruses according to the present invention,such as MVA, in particular MVA-BN and its derivatives, are useful forprime/boost administration. The viruses, in particular MVA strains suchas MVA-BN and its derivatives, as well as, corresponding recombinantviruses harboring heterologous sequences, can be used to efficientlyfirst prime, and then boost immune responses in naïve animals, as wellas, in animals with a pre-existing immunity to poxviruses. Thus, thevirus according to the present invention induces at least substantiallythe same level of immunity in vaccinia virus prime/vaccinia virus boostregimes compared to DNA-prime/vaccinia virus boost regimes.

A vaccinia virus, in particular an MVA strain, is regarded as inducingat least substantially the same level of immunity in vaccinia virusprime/vaccinia virus boost regimes when compared to DNA-prime/vacciniavirus boost regimes if the CTL response as measured in one of the “assay1” and “assay 2” as disclosed in U.S. Pat. No. 6,761,893, or even inboth assays, is at least substantially the same in vaccinia virusprime/vaccinia virus boost regimes when compared to DNA-prime/vacciniavirus boost regimes. The CTL response after vaccinia virusprime/vaccinia virus boost administration is higher in at least one ofthe assays, when compared to DNA-prime/vaccinia virus boost regimes. TheCTL response may be higher in both assays.

The virus used according to the present invention may be anon-recombinant virus, such as MVA, i.e., a virus that does not containheterologous nucleotide sequences. An example for a non-recombinantvaccinia virus is MVA-BN and its derivatives. Alternatively the virusmay be a recombinant virus, such as a recombinant MVA that containsadditional nucleotide sequences, which are heterologous to the virus.

The term “heterologous” as used in the present application refers to anycombination of nucleic acid sequences that is not normally foundintimately associated with the virus in nature; such virus is alsocalled “recombinant virus”.

The heterologous nucleic acid sequence may, for example, be selectedfrom a sequence coding for at least one antigen, antigenic epitope,beneficial proteins and/or therapeutic compound.

The term “beneficial proteins” as used in the present application refersto any proteins that are helpful in protecting an animal against anantigen selected from tumor antigen and foreign antigen, wherein thetumor antigen and the foreign antigen is different from the antigensassociated with the virus. Alternatively and more particularly the“beneficial proteins” are active in (i) increasing the level of factorswhich activate dendritic cells; and/or (i) increasing the number ofdendritic cells; and/or (iii) increasing the production and/or cellularcontent of an interferon. (IFN) or IL-12. Examples of such beneficialproteins are interferons, IL-12, Flt-3-L and or GM-CSF. Examples ofinterferons are type I interferons, such as IFN-alpha or IFN-beta.

The antigenic epitopes may be any epitope for which it is desired toinduce an immune response. Examples for antigenic epitopes are epitopesfrom Plasmodium falciparum, Mycobacteria, Influenza virus, from virusesselected of the family of Flaviviruses, Paramyxoviruses, Hepatitisviruses, Human immunodeficiency viruses or from viruses causinghemorrhagic fever such as Hantaviruses or Filoviruses, i.e., Ebola orMarburg virus. Thus, if e.g. a recombinant MVA expressing heterologousepitopes is used to vaccinate neonates according to the presentinvention, the result of this treatment is not only a generalvaccination due to the accelerated maturation of the immune system butalso a specific vaccination against the heterologous epitope expressedfrom the heterologous MVA.

A “therapeutic compound” encoded by the heterologous nucleic acid in therecombinant virus can be, e.g., a therapeutic nucleic acid such as anantisense nucleic acid or a peptide or protein with desired biologicalactivity.

The insertion of heterologous nucleic acid sequence may be done into anon-essential region of the virus genome. Alternatively, theheterologous nucleic acid sequence may be inserted at a naturallyoccurring deletion site of the viral genome (for MVA disclosed inPCT/EP96/02926). Methods how to insert heterologous sequences into theviral genome such as a poxyviral genome are known to a person skilled inthe art.

The present invention also provides a pharmaceutical composition and avaccine comprising a virus according to the present invention, such asMVA, e.g., for inducing an immune response in a living animal body,including a human.

The pharmaceutical composition may generally include one or morepharmaceutical acceptable and/or approved carriers, additives,antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Suchauxiliary substances can be water, saline, glycerol, ethanol, wetting oremulsifying agents, pH buffering substances, or the like. Suitablecarriers are typically large, slowly metabolized molecules such asproteins, polysaccharides, polylactic acids, polyglycollic acids,polymeric amino acids, amino acid copolymers, lipid aggregates, or thelike.

For the preparation of vaccines, the virus or its recombinants isconverted into a physiologically acceptable form. A person skilled inthe art is familiar with such methods. For MVA and other poxviruses thevaccine can be prepared based on the experience in the preparation ofpoxvirus vaccines used for vaccination against smallpox (as described byStickl, H. et al. [1974] Dtsch. med. Wschr. 99, 2386-2392). For example,the purified virus is stored at 80° C. with a titer of 5×10⁸ TCID₅O/mlformulated in about 10 mM Tris, 140 mM NaCl pH 7.4. For the preparationof vaccine shots, e.g., 10¹-10⁸ particles of the virus such as MVA arelyophilized in 100 ml of phosphate-buffered saline (PBS) in the presenceof 2% peptone and 1% human albumin in an ampoule, such as a glassampoule. Alternatively, the vaccine shots can be produced by stepwisefreeze-drying of the virus in a formulation. This formulation cancontain additional additives such as mannitol, dextran, sugar, glycine,lactose or polyvinylpyrrolidone or other additives such as antioxidantsor inert gas, stabilizers or recombinant proteins (e.g. human serumalbumin) suitable for in vivo administration. The glass ampoule is thensealed and can be stored between 4° C. and room temperature for severalmonths. However, as long as no need exists, the ampoule may be stored attemperatures below −20° C.

For vaccination or therapy the lyophilisate can be dissolved in 0.1 to0.5 ml of an aqueous solution, such as physiological saline or Trisbuffer, and administered either systemically or locally, i.e., byparenteral, intramuscular or any other path of administration know tothe skilled practitioner. The mode of administration, the dose, and thenumber of administrations, can be optimized by those skilled in the artin a known manner.

The virus according to the present invention, in particular MVA, can beadministered by oral, nasal, intramuscular, intravenous,intraperitoneal, intradermal, intra-utero and/or subcutanousapplication. In small animals the inoculation for immunization may begiven by parenteral or nasal administration; whereas, in larger animalsor humans, a subcutaneous, intramuscular or oral inoculation may beselected.

By way of example MVA may be administered in a dose of 10¹ TCID₅₀(tissue culture infectious dose) to 10⁹ TCID₅₀.

As indicated above, the virus according to the present invention, inparticular MVA, such as MVA-BN and its derivatives may be administeredin a therapeutically effective amount in a first inoculation (“priminginoculation”) and in a second inoculation (“boosting inoculation”).

In the context of the present invention the term “animal” also includeshuman beings. More generally, the animal is a vertebrate animal, such asa mammalian animal including a human. Specific examples of animals arepets, such as dogs and cats; economically important animals, such ascalves, cattle, sheep, goats, horses, pigs; and other animal such asmice, rats. MVA and DISC-HSV are particularly preferred viruses forthese animal species, and humans. The invention may also be used foreconomically important birds such as turkeys, ducks, goose and hens ifthe viruses used are capable of infecting avian cells, but not capableof being replicated to infectious progeny virus in said cells.

The term “domestic animals” as used in the present description refersinter alia to mammalian domestic animals, such as to dogs, cats, calves,cattle, sheep, goat, pigs, horses and deer.

According to a first alternative, the viruses according to the presentinvention, in particular MVA-BN and its derivatives may be used asspecific vaccines, i.e. to elicit an immune response that protects thevaccinated newborn against diseases caused by a virulent virus belongingto the same virus group, family or genus than the virus that was usedfor vaccination. By way of example MVA as defined above, in particularMVA-BN and its derivatives can be used to vaccinate newborn humansagainst poxvirus infections, in particular against smallpox. MVA, inparticular MVA-BN and its derivatives, may also be used to vaccinatevertebrate animals against poxvirus infections of veterinary importance.According to this first alternative the virus used for vaccination maybe a non-recombinant virus, such as MVA-BN or its derivatives, or arecombinant virus harboring genes in the viral genome that are notnaturally found in said genome. The recombinant virus may harboradditional genes that are helpful in stimulating the immune response.Examples for this kind of genes are cytokine genes and interferon genes.

According to a second, but related alternative, neonates are vaccinatedwith a recombinant virus harboring a heterologous nucleic acid sequence,as defined above, to induce an immune response against the amino acidsequence expressed from the heterologous nucleic acid sequence. By wayof example the nucleic acid sequence may code for an antigen or anantigenic epitope, as defined above. Examples for a recombinant virusaccording to this embodiment are recombinant MVA, in particularrecombinant MVA-BN or a derivative thereof, comprising a heterologousnucleic acid coding for antigens from (i) viruses other than MVA, suchas HIV-1, HIV-2, Denguevirus, West-Nile Virus, Japanese Enchephalitisvirus, measles virus, (ii) tumor antigens, (iii) bacteria, (iv) fungi.If the antigen expressed from the recombinant virus is, e.g., an HIVantigen it is possible to use the recombinant virus to induce an immuneresponse in the vaccinated neonate against HIV and to prevent AIDS. In abroader sense the recombinant virus expressing the antigen or antigenicepitope is used to induce an immune response against the agent fromwhich the heterologous sequence is derived and/or against the agent thatcomprises the antigen or antigenic epitope.

According to a third alternative, it has been unexpectedly found thatviruses that are capable of infecting the cells of the neonatal orprenatal animal, including a human, but not capable of being replicatedto infectious progeny virus in the neonatal or prenatal animal,including a human, can be used for the preparation of a medicament forprotecting an animal, in particular a newborn animal, including a human,against an antigen selected from tumor antigens and foreign antigens,wherein the tumor antigen and/or the foreign antigen are different fromthe antigens associated with the virus.

According to this third alternative, newborns vaccinated with theviruses according to the present invention, in particular with MVA, suchas MVA-BN and its derivatives, are protected against a challenge withforeign antigens such as infectious agents. Thus, the viruses accordingto the present invention, in particular MVA, are a general vaccine fornewborns. That is, by vaccinating newborns with the viruses according tothe present invention, in particular MVA, the immune system of thenewborns becomes more competent to deal with foreign antigens such asviruses. In the Example section, this is exemplified for vaccinationwith MVA and a subsequent challenge with Herpes simplex virus type 1.Thus, if the virus according to the present invention, in particularMVA, is used for the vaccination of newborns the vaccinated animals aremore protected against foreign antigens than unvaccinated animals duringthe critical time span until a functional and mature immune system isestablished.

According to the present invention “the tumor antigen and/or the foreignantigen is different from the antigens associated with virus”. This termis to be interpreted, in that according to this embodiment, theinvention is not primarily intended to use a virus, such as MVA, toinduce an immune response against the virus itself. Instead, the virusis used to induce a immune response, or at least a general immunestimulation, that protects the host against foreign antigens and tumorantigens, respectively, that are not associated with the virus. The term“antigens associated with the virus” refers to epitopes and antigens ofthe virus particle, and to antigens and epitopes on the surface of acell infected with the virus that are the result of the expression ofthe viral genome.

In the context of this embodiment the term “foreign antigens” refers toany antigens and epitopes that are not naturally a part, or a component,of the animal body. Foreign antigens are especially antigens andepitopes from infectious agents and toxins. Typical infectious agentsare viruses such as herpesviruses, retroviruses, rabiesviruses,rhabdoviruses, adenoviruses; bacteria such as Salmonella, Mycoplasm,Meningicoccus, Hemophilus; prions or fungi.

The invention is not only of interest to vaccinate animals againstforeign antigens but, in an alternative embodiment, is also suitable tovaccinate against tumor antigens. “Tumor antigens” are antigensassociated with certain tumoral diseases. Tumor antigens are most oftenantigens encoded by the genome of the host that develops the tumor.Thus, in a strict sense tumor antigens are not foreign antigens.However, tumor antigens are found in significant amounts in tumors;whereas, the amount of tumor antigens in normal tissues is significantlylower, and most often no tumor antigens are found at all in normaltissue. Examples for tumor antigens are known to the person skilled inthe art and include, e.g. the MAGE antigens. MVA is effective againstthese tumor antigens since the vaccination of an animal leads to anactivation and/or accelerated maturation of the immune system which thenmay lead to the destruction of tumor cells.

The term “protecting against an antigen” refers to the development of animmune response, which is directed against the foreign or tumor antigen.If the foreign antigen is an infectious agent, the host is protectedagainst the agent, i.e., the host develops an immune response againstsaid antigen. Consequently, the infection with the infectious agentleads to a less severe disease or to no disease at all. The term“protecting” is not to be understood in the sense that there is always a100% protection against the foreign or tumor antigen. Instead, the term“protection” as used in the present application refers to any beneficialeffect that helps the animal to deal with the foreign antigen and thetumor antigen, respectively.

According to the present invention such a protective effect is exertedfor at least 5 days, e.g., for at least 7, 14 or 28 days after the firstvaccination. In other words, the vaccinated and/or treated animal isprotected, e.g., against a foreign antigen if the animal comes intocontact with said antigen after 5, 7, 14 and 28 days, respectively.

In the context of the present invention the effect of the vaccination ofnewborns with the virus according to the present invention, inparticular with MVA may be explained by the induction or enhancement ofmaturation of the immune system and/or the activation of the immunesystem. In the context of the present invention, the term “induction orenhancement of the maturation of the immune system” refers inter alia tothe accelerated increase of dendritic cells or their precursors invaccines relative to controls. The terms “acceleration of thematuration” of the immune system and “enhancement of the maturation” ofthe immune system are used interchangeably in this description.

The “activation of the immune system” is characterized by the secretionand/or cell surface expression of molecules and hormones that facilitatecell/cell interaction or trafficking. Specific receptors take up thesesignals and respond. Activation markers are inter alia Flt3-L, IL-12,IFN-alpha, MHC-11 and CD8, in particular CD8alpha (see below).

The accelerated development/maturation of the immune system iscorrelated with (i) an increase of the level of factors activating andor mobilizing dendritic cells (DC) or their precursor cells; and/or (ii)an increase in the number of dendritic cells and their precursor cells;and/or (iii) an increase in the production and/or cellular content of aninterferon or IL-12. An example for DC precursor cells that are inducedby the virus according to the present invention, in particular by MVA,are plasmacytoid DC precursors that are very important for the defenceagainst viral infections and that seem to produce IFN α/β.

More specifically, the enhancement of the maturation of the immunesystem may be defined by an at least 2-fold increase in surface markersfound on DC, such as MHC-II, CD40 and/or CD80/86. Such an increase canbe measured in the blood. Additional markers to characterize anenhancement of the maturation of the immune system are Flt3-L, IL-12,IFN-alpha, MHC-II and CD8a (see below). Moreover, the acceleratedmaturation of the immune system may be correlated to an at least 1.5fold increase, such as at least a 2.0-fold increase in the number ofCD11c positive cells in the blood, and/or the spleen, 7 days after theadministration of MVA-BN to newborn animals when compared to controlanimals that have not received MVA-BN. Moreover, the enhancement ofmaturation of the immune system may be correlated with at least a1.5-fold increase, such as at least a 2.0-fold increase of theconcentration of Flt3-L, two days after the vaccination of neonates withviruses according to the present invention, when compared to age matchedcontrols.

In this context it is to be noted that there is an association betweenthe phenotype and function of murine and human DC populations that canbe characterized by their surface phenotype (Hochrein et al. 2002. Hum.Immunol. 63: 1103). Dendritic cells in the blood can be detected usingflow cytometry using a range of surface markers (MacDonald et al. 2002.Blood. 100:4512) that also allow specific populations of DC, such as theplasmacytoid DC to be identified (Dzionek et al. 2002. Hum Immunol. 63:1133; Dzionek et al 2000. J. Immunol. 165: 6037). Using similartechniques DC can also be detected in other human tissues (Summers etal. 2001. Am. J. Pathol. 159: 285).

According to the present invention the viruses as defined above mightalso be used to treat neonatal or prenatal animals to (i) increase thelevel of factors activating and or mobilizing dendritic cells (DC) ortheir precursor cells; and/or (ii) to increase in the number ofdendritic cells and their precursor cells; and/or (iii) to increase inthe production and/or cellular content of an interferon or IL-12. It hasbeen demonstrated that following vaccination with MVA-BN theplasmacytoid dendritic cells upregulate MHC-11 and CD8a and producesignificantly more IL-12 and IFN-alpha. The increase of IL-12 after theadministration of the viruses used according to the present inventionmay be 2-fold, 100-fold, 500-fold, 1000-fold, 2500-fold or 5000-fold.The increase of the concentration of Flt3-L two days after thevaccination of neonates with viruses according to the present invention,such as MVA-BN or its derivatives, may be at least 1.5-fold, such as atleast 2.0-fold when compared to age matched controls.

The term “activation of dendritic cells or their precursors” refers tothe maturation of DC to antigen presenting cells through ill-definedcell stages driven by hormones and different antigenic stimuli.Intermediates of DC are termed precursors. These immature DC reach theperiphery. Activation markers which are upreguated in activateddendritic cells are inter alia Flt3-L, IL-12, IFN-alpha, MHC-11 and CD8a(see below).

It was noted that hormones such as GM-CSF lead to more immature DC inthe periphery. Because GM-CSF leads to more DC precursor in bone marrow,blood and peripheral organs (and the cells have to move there), thisphenomenon has been termed “mobilization of dendritic cells or theirprecursors”. This definition is also used in the present description.

Consequently, the vaccination of animals including a human is especiallyuseful, if it is intended to (i) increase the level of factorsactivating dendritic cells (DC) or their precursor cells; and/or (ii)increase the number of dendritic cells or their precursor cells; and/or(iii) increase the production and/or cellular content of an interferonor IL-12.

Factors that activate dendritic cells include inter alia Flt3-L (Lymanet al., Cell 1993, 75: 1157-1167) and GM-CSF. Typical interferonsaccording to the present invention are IFN-alpha and IFN-beta. Theviruses used according to the present invention induce Flt3-L and it isassumed that some of the beneficial effects observed are due to saidinduction.

In the context of the present application a newborn animal, or human, isdefined as an animal or human, not yet having a mature immune system.Throughout this specification the terms “newborn animal” and “neonatalanimal” are used synonymously. A mature immune system is characterizedby the ability to fully activate the innate immune system, and by thefact that all known T and B cell functions and products are in place; inparticular immunoglobulin isotypes such as IgA and IgE. Thus an immatureimmune system is characterized by a low number of T cells, B cells anddendritic cells relative to adults; by low IFN production compared toadults; and by the fact that the secondary lymphoid organs are not fullymature. More specifically a “neonatal” or “newborn” in the context ofthe present invention may be defined as an infant animal having a numberof splenic CD4+ cells being at least 2-fold, at least 20-fold, at least200-fold, at least 2,000-fold, or even at least 20,000-fold lower thanthe average number of splenic CD4+ cells in adults.

In mice the immune system is mature at the age of 4 weeks. In humansmaturity is probably 6 months to 1 year. In cats and dogs the immunesystem is mature usually at the age of 6 months; in calves, sheep andpigs at the age of 4-12 weeks. Vaccination with the virus according tothe present invention, in particular with MVA, may be done during beforethe immune system is mature. However, since maturity develops almostexponentially after birth, it is possible to vaccinate with the virusaccording to the present invention, in particular with MVA, as earlyafter birth as possible. Since in all relevant domestic animals, and inhumans, the immune system is mature not earlier than 4 weeks afterbirth, the vaccination with the virus according to the presentinvention, in particular with MVA, is done within 4 weeks after birth,e.g., within 2 weeks after birth, within 1 week after birth or evenwithin 3 days after birth. These general terms are applicable to allimportant domestic animals, in particular to all important domesticmammalian animals, including humans. The person skilled in the art willbe aware of the fact that even older animals may be regarded asnewborns/neonates in the context of the present invention; and thereforevaccination may also be successfully carried out with older animals,when the immune system is not yet mature 4 weeks after birth. Thus, inhumans the vaccination may be carried out within 6 months after birth,e.g., within 3 months after birth, within 2 months after birth, within 4weeks after birth, within 2 weeks after birth, within 1 week after birthor even within 3 days after birth.

Since the best effects of the virus according to the present invention,in particular MVA as a general vaccine are observed if the virus isadministered to an immature immune system, it might be useful tovaccinate even prenatal animals including humans. Prenatal vaccinationmay be desirable in economically important animals such as cattle orpigs. If the placenta allows the passage of the virus, the prenate canbe vaccinated simply by vaccinating the mother animal. Thus, thevaccination of the mother animal to vaccinate the prenate isparticularly promising in an animal having a placentaendotheliochorialis, such as dogs, cats, rats and mice or having aplacenta heamochorialis, such as primates including humans. In animalshaving a placenta chorionepithelialis, such as cattle and sheep orhaving a placenta syndesmochorialis, such as pigs and horses, thevaccination of prenates can be done by in utero administration. Ofcourse, this mode of administration can be also done for animal having aplacenta endotheliochorialis or haemochorialis.

Since the viruses according to the present invention, in particular MVA,lead to an accelerated maturation of the immune system and are, thus,useful as a general vaccine, the vaccinated animals are protectedagainst a variety of diseases. More specifically the viruses accordingto the present invention, in particular MVA, can be used to vaccinatecats for general well being and against feline herpes or felineinfectious peritonitis. The viruses according to the present invention,in particular MVA, may be used in dogs for general well being andagainst respiratory tract associated (viral) diseases. The virusesaccording to the present invention, in particular MVA, may be used inpigs for general well being and against Hemophilus or Mycoplasminfections, especially in fattening pigs.

As previously indicated, one may administer the viruses according to thepresent invention, in particular MVA, in newborns or prenatal animals toprotect said animal against a foreign antigen and/or a tumor antigen,wherein the tumor antigen is different from the antigens associated withthe virus used for vaccination. However this embodiment is notrestricted to newborn and prenatal animals. Instead, in an alternativeembodiment, the invention can be carried out for animals of all ages,since a beneficial effect can be observed also in adult animals. Thus,according to this embodiment the viruses as defined above, in particularMVA-BN and its derivatives, are useful to protect an animal, including ahuman, against an antigen selected from tumor antigen and foreignantigen, wherein the tumor antigen and/or the foreign antigen isdifferent from the antigens associated with the virus. As indicatedabove, the viruses used according to the present invention are capableof infecting cells of the animal but not capable of being replicated toinfectious progeny virus in said cells. All information, definitions,including the definition of the duration of the protective effect, alsoapply for the present embodiment according to which the virus may alsobe administered to adults.

In contrast to newborns, the immune system of adult animals has alreadymatured. Nevertheless, it might be that the immune system is weakeneddue to certain diseases or simply due to the age of the animal.Especially in immune-compromised people and in elderly people, theadministration of the viruses according to the present invention, inparticular MVA, to the animal may have a beneficial effect inter alia by(i) increasing the level of factors activating and/or mobilizingdendritic cells (DC) or their precursor cells; and/or (ii) by increasingthe number of dendritic cells or their precursor cells; and/or (iii) byincreasing the production and/or cellular content of an interferon orIL-12. Thus, even in adult animals, the administration of the virusesaccording to the present invention, in particular MVA, may lead to anincreased competence of the immune system to deal with foreign antigensand/or tumor antigens. In other words, the viruses used according to thepresent invention are useful for the activation of the immune system ingeneral.

The invention further concerns the viruses according to the presentinvention, in particular MVA, for the preparation of a medicament to beadministered to an animal, including a human, wherein said medicament(i) increases the level of factors which activate dendritic cells;and/or (ii) increases the number of dendritic cells; and/or (iii)increases the production and/or cellular content of an interferon (IFN)or IL-12. All definitions given above for the other embodiments are alsoapplicable for the present embodiment. According to this embodiment theinvention does not aim primarily at inducing a protection againstforeign antigens and/or tumor antigens. Instead, this embodiment isaimed at treating conditions and diseases characterized by (i) a lowlevel of factors which activate dendritic cells; and/or (ii)insufficient or too low number of dendritic cells; and/or (iii) lowproduction and/or cellular content of an interferon (IFN) or IL-12.Thus, according to this embodiment the treatment with the virusesaccording to the present invention, in particular MVA could protectagainst allergies or autoimmune diseases. Again this treatment isparticularly promising if the viruses according to the presentinvention, in particular MVA, are administered to newborn animals.

Additionally, according to a further embodiment the virus according tothe present invention, such as MVA, in particular MVA-BN and itsderivatives, is particularly useful to induce immune responses inimmuno-compromised animals, e.g., monkeys (CD4<400/μl of blood) infectedwith SIV, or in immuno-compromised humans. The term “immuno-compromised”describes the status of the immune system of an individual, which showsonly incomplete immune responses or has a reduced efficiency in thedefence against infectious agents.

The invention further concerns a method for protecting an animal,including a human, against an antigen selected from tumor antigen andforeign antigen, by administration of a virus according to the presentinvention, in particular Modified Vaccinia virus Ankara (MVA), whereinthe tumor antigen and/or the foreign antigen is different from theantigens associated with the virus.

In a further embodiment the invention concerns a method for thetreatment of an animal, including a human, comprising the administrationof a Modified Vaccinia virus Ankara (MVA) to (i) increase the level offactors which activate dendritic cells; and/or (ii) increase the numberof dendrite cells; and/or (iii) increase the production and/or cellularcontent of an interferon (IFN) or IL-12.

According to an alternative embodiment of the present invention, one ormore of the beneficial proteins as defined above, such as type IInterferon and/or Flt-3L may by administered to the animal instead of,or in addition to, the virus that is capable of infecting cells of theanimal but not capable of being replicated to infectious progeny virusin the cells. The effect that is achieved by administering one or moreof the beneficial proteins such as type I Interferon and/or Flt-3L,optionally in combination with the virus a defined above, is comparableto that achieved if the virus, as defined above, is administered alone.Thus, the effect may be (i) a long-term protection of the animal againstforeign antigens and tumor antigens; and/or (ii) a long-term increase ofthe number of dendritic cells in the animal; and/or (iii) an inductionor enhancement of the maturation of the immune system of the animal;and/or (iv) an activation of the immune system of the animal.

If not stated otherwise, all definitions given so far are alsoapplicable for the alternative embodiment. In the absence of indicationsto the contrary, the term “animal” generally also covers humans, i.e.,the animal may be a human.

According to one example of this embodiment, it is sufficient toadminister to the animal, including a human, one or more of thebeneficial proteins, and it is not necessary to administer to theanimal, including a human, a virus that is capable of infecting cells ofthe animal but is not capable of being replicated to infectious progenyvirus in the cells.

According to another example of this embodiment, it is possible toadminister to the animal, including a human, one or more of thebeneficial proteins together with a virus that is capable of infectingcells of the animal but not capable of being replicated to infectiousprogeny virus in the cells. The administration of one or more beneficialproteins and the virus may be made simultaneously or within a timeinterval, e.g., the beneficial protein(s) may be administered before orafter the administration of the virus. The time interval may be anyconvenient interval that leads to a beneficial effect in the animal,including a human. By way of example, the time interval between theadministration of the beneficial protein(s) and the virus may be in therange of 1 day-2 months, such as in a range of 2 days-1 month, or in arange of 3 days-2 weeks, or the time interval may, for example, be about1 week. It is possible to first administer the beneficial protein(s) andthen to administer the virus. Alternatively, it is also possible tofirst administer the virus and then to administer the beneficialprotein(s). The virus used in this example of the alternative embodimentmay be a DNA virus. The DNA virus may be a DISC-Herpesvirus or aModified Vaccinia virus Ankara (MVA). The MVA strain may be MVA-BN,deposited at the European Collection of Animal Cell Cultures (ECACC)with the deposition number V00083008, and derivatives thereof. The virusmay be a recombinant virus, the genome of which comprises at least oneheterologous nucleic acid.

The type I interferon may be IFN-alpha or IFN-beta. Any IFN-alpha or-beta may be used. The term “IFN-alpha” includes naturally occurringIFN-alpha, recombinant IFN-alpha, synthetic IFN-alpha, consensusIFN-alpha, modified IFN-alpha and derivatives of IFN-alpha, as well asfusion proteins comprising an IFN-alpha moiety. The term “IFN-beta”includes naturally occurring IFN-beta, modified IFN-beta, recombinantIFN-beta and derivatives of IFN-beta, as well as fusion proteinscomprising an IFN-beta moiety. Non-limiting examples of IFN which isnon-species specific are recombinant hybrid interferon hIFN (Gehring etal., Journal of Medical Virology 2005, 75:249-257) and alpha 2a-IFN(Parez, N. et al, British Journal of Haematology, 2000, 110, 420-423).

The term “modified” Interferon relates to Inferferons being modifiedwith non-protein residues. Examples of modified IFN are PEG(polyethylene glycol) modified IFN (Gehring et al., Journal of MedicalVirology 2005, 75:249-257; Pawlotsky, J.-M., N. Engl. J. Med. 2004,351:422-423) or glycosylated IFN.

The term “derivative” of an Interferon relates to proteins in which oneor more amino acids are deleted, substituted, modified or inserted withrespect to known Interferon protein sequences. The Interferonderivatives according to the present invention may be derivatives that(i) still have the biological activity of inducing a long-termprotection in an animal against foreign antigens and tumor antigens;and/or (ii) still have the biological activity of long-term increasingthe number of dendritic cells; and/or (iii) still have the biologicalfunction of inducing or enhancing the maturation and/or activation ofthe immune system of an animal.

IFN is administered in concentrations that depend on the species towhich it is to be administered and the kind of IFN. Typical standardconcentrations are known to the person skilled in the art. IFN may beadministered once.

Alternatively, it is also possible to administer IFN several times,e.g., once a week for several weeks; or every 2 to 4 days for one toseveral weeks. By way of example and without being bound thereto,reference is made to the following administration schemes: Mice may betreated with hIFN ranging in concentration from 1×10³ to 1×10⁵U/injection. Human PEG-IFN-alpha may be administered to mice in weeklydoses of 0.1 to 10 μg. In humans, including infants, alpha2a-IFN may beadministered, e.g., in a concentration of 1-3×10⁶ per injection, e.g., 1to 3 times a week. In humans, PEG-IFN may be administered in a weeklydose of about 180 μg per week (Marcellin, P. et al., N. Engl. J. Med.2004, 351:1206-1217)

Detailed information regarding Flt-3L, derivatives of Flt-3L such astruncated forms, dosages and modes of administration are given innumerous documents known to the person skilled in the art, e.g., in U.S.Pat. No. 6,190,655. Flt-3L may be a recombinant Flt-3L and/or a modifiedFlt-3L, such as PEG (polyethylene glycol) modified Flt-3L.

The actual dosage of Flt-3L depends on the species to which it is to beadministered. Flt-3L may be administered once. Alternatively, it is alsopossible to administer Flt-3L several times, in the same regimesdescribed above for IFN.

It is possible to administer only one factor, selected from type Iinterferon and Flt-3L. Alternatively, it is also possible to add two ormore type I interferons, such as IFN-alpha and IFN-beta, or to add oneor more type I Interferons and Flt-3L.

The effect of protecting an animal, including a human, as well as, theeffect of increasing the number of dendritic cells, is a long-termeffect. The effect is exerted, for example, for at least 5 days, for atleast 7 days, for at least 14 days or for at least 28 days after theadministration of the one or more beneficial proteins, such as a type IInterferon and/or Flt-3L.

The term “foreign antigen” in the context of the use of the beneficialproteins, refers to any antigens and epitopes that are not naturally apart or a component of the animal body (see also the definition givenabove). The “foreign antigen” may be any infectious agent such as abacterium, a prion, a parasitic agent, a eukaryotic unicellular ormulticellular infectious agent, a fungus or a virus. The virus may beany virus, such as poxvirus, smallpox virus, herpes virus, retrovirus,HIV, measles virus, rubella virus, rhinovirus, yellow fever virus,dengue virus, hepatitis viruses A, B, or C, rabies virus, rhabdovirus orany other virus.

The term “tumor antigen” has been defined above.

The animal may be a neonatal or prenatal animal, as defined above, inparticular if it is intended (i) to protect the animal against foreignantigens and tumor antigens; (ii) to increase of the number of dendriticcells in the animal; (iii) to induce or enhance the maturation of theimmune system; and/or (iv) to activate the immune system of the animal.

The animal may also be an adult animal, in particular if is intended (i)to protect the animal against foreign antigens and tumor antigens; (ii)to increase of the number of dendritic cells in the animal; and/or (iii)to activate the immune system of the animal.

Thus, according to this embodiment, the method according the presentinvention is useful for animals, including humans, of all age groups,including adults and elderly people as far as humans are concerned.

The animal may also be an immune-compromised animal.

The invention further concerns one or more beneficial proteins,optionally in combination with a virus that is capable of infectingcells of the animal but not capable of being replicated to infectiousprogeny virus in the cells, which is administered for a general immunestimulation. Thus, by way of example, this embodiment refers to theadministration of one or more of the beneficial proteins, such as type IInterferon and/or Flt-3L to an animal, including a human, to protectsaid animal, including a human, for at least 5 days against foreignantigens, such as viruses.

There are many circumstances under which this embodiment is ofparticular interest, e.g., the one or more beneficial proteins,optionally in combination with a virus that is capable of infectingcells of the animal but not capable of being replicated to infectiousprogeny virus in said cells, may be administered to protect humansagainst diseases for which no vaccine is available. The one or morebeneficial proteins, optionally in combination with a virus that iscapable of infecting cells of the animal but not capable of beingreplicated to infectious progeny virus in said cells, also can beadministered to protect humans against infectious diseases if there isnot enough time for a vaccine to induce a protective immune response.This can be the case if one has to travel immediately to a country inwhich infectious diseases are endemic.

As discussed previously, the alternative embodiment may relate interalia to the administration of beneficial proteins as defined above, suchas type I Interferon and/or Flt-3L, optionally in combination with avirus as defined above, for a long-term increase of the number ofdendritic cells in the animal, including a human. The dendritic cells(DC) are selected from conventional DC (cDC) and plasmacytoid pre DC(pDC). Methods are known to the person skilled in the art as to how anincrease in the number of dendritic cells can be determined and as tohow cDC's can be distinguished from pDC's (see O'Keeffe, M. et al., J.Exp. Med. 2002, 196:1307-1319).

The invention further relates to a composition comprising a combinationof two or more of the beneficial factors as defined above, e.g.,IFN-alpha and IFN-beta; IFN-alpha and Flt-3L; IFN-beta and Flt-3L;IFN-alpha, IFN-beta and Flt-3L.

The invention further relates to a composition comprising a combinationof one or more of the beneficial proteins, as defined above, and a virusthat is capable of infecting cells of the animal but not capable ofbeing replicated to infectious progeny virus in said cells, and which isadministered to the animal, including a human. By way of example, such acombination may comprise: IFN-alpha and MVA-BN or a derivative thereof;IFN-beta and MVA-BN or a derivative thereof; Flt-3L and MVA-BN or aderivative thereof; IFN-alpha, Flt-3L and MVA-BN or a derivativethereof; IFN-beta, Flt-3L and MVA-BN or a derivative thereof; IFN-alpha,IFN-beta, Flt-3L and MVA-BN or a derivative thereof; or any othercombination.

The invention further relates to a kit comprising (i) one or morefactors as defined above and a virus as defined above or (ii) two ormore factors as defined above and, optionally, a virus as defined above;wherein the kit comprises at least two vials, and wherein the vialscomprise different factors, combinations of factors, and/or viruses.Thus, one vial may comprise IFN-alpha and a second vial comprises MVA-BNor a derivative thereof; one vial may comprise IFN-beta and a secondvial comprises MVA-BN or a derivative thereof; one vial comprises Flt-3Land a second vial may comprise MVA-BN or a derivative thereof; one vialmay comprise IFN-alpha, a second vial may comprise Flt-3L and a thirdvial may comprise MVA-BN or a derivative thereof; one vial may compriseIFN-beta, a second vial may comprise Flt-3L and a third vial maycomprise MVA-BN or a derivative thereof; one vial may compriseIFN-alpha, a second vial may comprise IFN-beta, a third vial maycomprise Flt-3L and a fourth vial may comprise MVA-BN or a derivativethereof; or any other combination. It is also within the scope of theinvention, that one of the at least two vials comprises a combination oftwo or more factors as defined above; or a combination of one or morefactors, as defined above, and a virus, as defined above, and whereinthe second vial comprises only one factor or virus, as defined above, ora combination of factors and/or virus that is different from thecombination in the first vial.

According to a further alternative embodiment, it is also possible toadminister dendritic cells from an animal that was treated with afactor, as defined above, or a virus, as defined above, to anotheranimal to protect the animal against foreign antigens and/or tumorantigens. The factor may be selected from type I Interferons and Flt-3L,and the virus may be a MVA strain, such as MVA-BN. All definitions,concentrations and combinations given above apply also to this furtherembodiment. The DC's may be CD11+ cells. Methods are known to the personskilled in the art as to how to obtain DC and CD11+ cells, respectively.The first animal may be an animal of any age group, e.g., a neonatalanimal. The cells may be isolated 5 days, 7, 8, 14 or even 28 days afterthe administration of the one or more beneficial proteins, such as atype I Interferon and/or Flt-3L, and/or the virus, as defined above.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1A: Newborn mice were injected once within 24-48 h of birth with106 p.f.u. of MVA or DISC HSV-1 or treated with physiological saline(NaCl) as controls. At 7 days of age, CD11c, a pan DC marker was used todetermine these cells in peripheral blood by flow cytometry. The meanand standard deviation of 3 to 5 experiments are shown.

FIG. 1B: The experiment was conducted as indicated for FIG. 1A. However,CD11c cells were determined in spleen by flow cytometry.

FIG. 1C: The experiment was conducted as indicated for FIG. 1A. However,CD11c cells were determined in peritoneal fluid by flow cytometry.

FIG. 2: Mice were vaccinated with MVA-BN as indicated in the leftcolumn. After two weeks, the percentage of CD11c⁺ single and CD11c⁺/CD8⁺double positive cells in spleen and in blood were determined by flowcytometry.

FIG. 3: Newborn mice were injected with MVA or NaCl, as a control, atday one and 5 of age. At day 8, murine Flt3-L was determined in serum ofthese mice by ELISA and the values are given as pg/ml.

FIG. 4: Newborn mice were injected once within 24-48 h of birth with 10⁶p.f.u. of MVA or treated with NaCl, as controls. At 7 days of age, allmice were exposed to 100×LD₅₀ of HSV-1 strain F. The number of survivinganimals was monitored for 21 days.

FIG. 5: Mice were treated as indicated for FIG. 4. The data represent 9different challenge experiments with 100 LD₅₀ of HSV-1. None of thecontrol animals survived the challenge.

FIG. 6: Survival of adult mice vaccinated on the first day of life withMVA-BN following a lethal vaccinia challenge. Three litters of 6,1-day-old pups (18 mice) were vaccinated with MVA-BN (2.5×10⁷ TCID₅₀)and at 4 weeks (adult mice) challenged with a lethal dose of vaccinia.MVA-BN vaccination clearly induced a protective immunity in neonatalmice that lasted until adulthood.

EXAMPLES

The present invention will be better understood in connection with thefollowing examples, which are intended as an illustration of and not alimitation upon the scope of the invention.

Example 1 (i) MVA-BN and DISC-HSV Induces DC of the CD11c⁺ and CD8⁺Phenotype in Newborn Animals

First set of experiments: Newborn mice are naturally immunodeficientbecause the IFN system is not mature. Dendritic cells can be induced invitro, as well as in vivo, by a variety of stimuli. The number andactivation state of DC, the best producers of IFN, is evaluated usingflow cytometry. In these studies it was determined whether a controlledMVA-BN replication could induce DC, which is evaluated phenotypically.Groups of mice were injected with 10⁶ plaque forming units (p.f.u.) ofMVA-BN or saline within 1-2 days after birth, and in some cases 5 daysafter birth. Blood and spleen cells from individual mice of both groupswere analyzed by FACS and the data compared.

Data from 7 to 8 individual mice indicate that treatment with MVA-BNincreased CD11c⁺ cells 2-3 fold, accompanied with increased expressionof MHC II and increased presence of T cells of the CD4 or CD8 type.Interestingly, CD19/54, a marker for mature B cells, decreasedindicating that these cells either emigrated in organs other than spleenor that precursors of B cells were recruited early to other lineages,notably DC of the plasmacytoid phenotype that carries early B cellmarkers (B220).

Data from three different experiments indicated reproducibility andsignificant differences. Experiments with DISC-HSV-1, a differentreplication controlled viral vaccine, demonstrates the induction ofsimilar amounts of CD11c+ cells after neonatal priming.

The results are summarized in FIG. 1A-C.

To further investigate subpopulations of DC in blood and spleen, andanalyze the long-term effect of treatment with MVA-BN, cells in bloodand spleen were analyzed at 2 weeks of age. At this time point, treatedanimals had about twice the number of CD11c⁺ cells in spleen than theamount observed at one week of age. A single treatment with the virus atbirth, resulted in a 3-fold elevated number of these cells in spleen 2weeks later (FIG. 2). Similar effects were seen in blood, with theexception that the population of CD11c+/CD8a+ were about 4 times higher.A single treatment with MVA-BN at 7 days after birth, leads to anincrease of CD11c+/CD8a+, from 13- to 40-fold, with a less dramaticeffect on the CD11c⁺ cells. As expected, two vaccinations, at birth andat day 7, had a significant effect on the population of CD11c⁺ cells.The various effects are shown in FIG. 2.

In a second set of experiments, one-week-old mice that were vaccinatedat birth with 2.5×10⁷ TCID₅₀ of MVA-BN showed a different composition ofimmunologically relevant cell populations in spleen and blood whencompared to control mice (Table 1). In blood, there was an increase inthe CD8 positive lymphocyte population, as well as an increase in thenumber of NK cells. The number of CD11c positive cells was about 3 timeshigher than in controls and the percent of B-cells (B220 and CD19 doublepositive cells) was significantly decreased. In the spleen, the totalnumber of cells did not differ between immunized animals and controls.In contrast to the blood, the spleen of vaccinated animals had more CD4positive T lymphocytes than controls and the number of NK cells was notincreased. Similar to blood, the relative number of CD8 positivelymphocytes was increased and the number of B-cells decreased. Thepercentage of CD11c positive cells was about 3 times higher than incontrols. A difference in the percentage of dendritic cells wasrecognized at day 5 following vaccination with MVA-BN, wherein thenumber of CD11c positive cells in the spleen of 4, untreated controlswas 3.6%, compared to 4.8% in the 4, MVA-BN vaccinated mice. The sameamount of UV-inactivated MVA-BN did not cause any significant change inthe cell populations after vaccination of neonatal mice compared tocontrols (data not shown). The initial vaccination dose was chosenarbitrarily. After titration of the inoculum, a standard dose of 2.5×10⁶TCID₅₀ was selected for vaccination (10 times less than in the initialexperiment). At this dose maximal numbers of DC were induced (Table 2).

TABLE 1 Changes induced in blood and spleen cells in newborn mice 1 weekafter immunization with 2.5 × 10⁷ TCID₅₀ MVA-BN Blood Spleen Parameter %NaCl MVA-BN P* NaCl MVA-BN P* Total cells × 10⁶ 17.9 ± 1.9  24.1 ± 2.6 0.105 % CD11c  5.4 ± 1.3 18.6 ± 1.5 0.001 2.8 ± 0.1 7.9 ± 0.8 0.001 %CD11c/CD8α  0.5 ± 0.1  2.7 ± 0.3 0.001 1.1 ± 0.1 4.6 ± 0.7 0.002 %CD4/CD3 16.9 ± 1.1 16.1 ± 1.5 0.999 4.8 ± 0.3 8.1 ± 1.5 0.004 % CD8α/CD3 6.0 ± 0.9 10.3 ± 0.9 0.002 4.7 ± 0.3 8.4 ± 1.1 0.002 % NK1.1/DX5 16.4 ±1.2 24.4 ± 3.3 0.032 2.5 ± 0.3 2.4 ± 0.2 0.862 % CD19/B220 22.3 ± 0.5 8.4 ± 0.8 0.001 16.2 ± 1.3  8.6 ± 0.9 0.004 *Mann-Whitney U-Test

TABLE 2 Induction of CD11c positive cells in the spleen within 7 daysafter MVA-treatment of 1-day-old wt mice or mice with gene-targeteddisruptions. MVA dose controls MVA-BN Mouse strain (TCID₅₀) % CD11c %CD11c ratio wt^(a) 2.5 × 10⁷ 2.8 7.9 2.8 wt 2.5 × 10⁶ 2.1 11.9 5.6 wt2.5 × 10⁵ 2.5 6.6 2.6 RAG^(b) 2.5 × 10⁷ 4.2 5.4 1.3 AG129^(c) 2.5 × 10³2.6 2.7 1.0 ^(a)Wt = either C57BL/6 or 129 Sv/Ev mice. ^(b)RAG micedeletion in recombination activating gene (i.e. no functional T and Bcells). ^(c)AG129 gene targeted disruptions of IFN receptor Type I(IFN-alpha and -beta) and Type II (IFN-gamma)

(ii) MVA-BN Induces Preferentially Plasmacytoid Dendritic Cells (pDC)

According to other authors CD11c positive cells that also expressedCD45RA or CD45R were considered as pDC (Asselin-Paturel, et al. 2001,Nat Immunol, 12: 1144). It was, therefore, determined whether MVA-BNinduced an increase of pDC. A further experiment was performed in whichalso CD45RA or CD45R on CD11c positive were analysed. The percentage ofCD11c and CD45R double positive cells was significantly higher in MVA-BNtreated mice (5.6±0.7%) than in both control groups (untreated 3.0±0.3%,p=0.01; UV-inactivated MVA-BN 3.0±0.2%, p=0.006. Mann-Whitney U-test).

(iii) Neonatal Mice Treated with MVA-BN have Elevated Levels of SerumFlt3-L

Flt3-L is a hematopoetic factor that leads to increased levels of DC inadult animals. In humans and possibly mice, the richest source of thisfactor are activated T cells. To determine whether the elevated numbersof DC could be the result of induced Flt3-L, serum of MVA-BN treatedmice was compared to mock treated animals for the presence of thisfactor. Animals treated at day 2 and 5 had twice the levels of Flt3-L inthe serum when compared to serum of mock treated animals. Hence, Flt3-Lis one of the factors that could be made responsible for elevatednumbers of DC (FIG. 3)

The time course of the Flt3-L induction in newborn mice was assessedafter administration of MVA-BN. In newborns, MVA-BN vaccination inducedan increase in Flt3-L concentration within 24 hours. The inductionreached a maximum after 48 hours and was still present at day 7, thetime when spleen cells were usually analyzed and resistance againstHSV-1 was tested (see below). In the vaccinated mice the Flt3-Lconcentration in the serum was two-fold increased 24 hours and 48 hoursafter the vaccination, compared with age matched control animals.

Role of MVA-BN-Induced Type I IFN in Resistance Against HSV-1 Challenge.

The ability of MVA-BN to induce IFN type I in newborn mice wasevaluated. Elevated levels of serum IFN-alpha was not detected inneonatal or 12-day-old mice 2 days after exposure to MVA-BN. Incontrast, cultures of pDC from 1 week old mice infected in vitro withMVA-BN at a multiplicity of infection of 1, secreted ˜1000-1500 U ofIFN-alpha/10⁵ cells. In uninfected cultures, no IFN-alpha was detected.Thus, MVA-BN was demonstrated to induce secretion of IFN-alpha in vitroand expansion of DC in vivo.

In an effort to determine whether the IFN type 1, which was inducedafter MVA-BN administration, increased protection, 10⁵ U of rIFN-alphaB/D was injected into naïve mice either once (at birth) or twice (atbirth and on the following day) and evaluated for the effect bychallenge with HSV-1. Five days after infection, all seven untreatedmice were dead. By contrast, six of the seven mice receiving only onetreatment, and all seven mice receiving two rIFN treatments, survivedfor five days. One of the seven mice that received one treatment withIFN, and three of the seven mice that received two treatments with IFN,were still alive at day 21, which means that they survived thechallenge.

Example 2 MVA-BN Treated Neonatal Mice Survive a Challenge with 100 to500 LD₅₀ of HSV-1

Groups of mice were treated with the standard dose of MVA-BN one or 2days after birth and challenged at 7-8 days of age with 100 to 500 LD₅₀of Herpes simplex virus 1 (HSV-1) (FIG. 4). MVA BN treated mice survivedthe challenge with HSV 1, whereas all the control mice died within 5-6days after inoculating the challenge virus.

To further support these observations, 9 challenge experiments wereperformed with 40 MVA BN treated and 45 control mice. More than 80% ofthe virus treated mice survived the challenge, whereas all the controlmice died (FIG. 5).

In a separate set of experiments the mice were treated at birth withMVA-BN (2.5×10⁶ TCID₅₀/mouse). At day 8 a challenge with either 10³ (1LD₅₀) or 10⁵ (100 LD₅₀) PFU of HSV-1 was performed. Following MVA-BNvaccination 65% of the mice survived a viral dose that killed 100% ofthe control mice (100 LD₅₀) and 90% survived a dose that killed 45.5% ofthe controls (1 LD₅₀). In additional experiments a group of 7 micevaccinated with UV-inactivated MVA-BN were infected with HSV-1. Five ofthem died within 7 days. The remaining 2 animals ceased to grow and diedat day 22 and 29. Therefore, mice treated with MVA-BN reached a state ofincreased resistance against HSV-1 that was associated with live MVA-BN,but not UV-inactivated MVA-BN.

In control experiments done with mice that do not have functionalT-cells it was determined that the protection against HSV-1 aftervaccination with MVA-BN was not due to cross-reacting cytotoxicT-lymphocytes induced by MVA-BN.

It was tested whether DC cells were responsible for the protection ofmice from HSV-1 after vaccination with MVA-BN. To this end, naïve8-day-old mice were challenged with 5×10⁴ PFU HSV-1 4 hr after transferof cells from MVA-treated mice. In a first experiment splenocytes from8-day-old mice treated at 1 day of life with MVA-BN were separated in DCrich (low-density) and DC poor (high-density) fractions. Mice receiving5×10⁶ cells from the DC rich fraction survived the challenge to 50%whereas all the mice receiving 10 times less DC rich suspension oruntreated mice died within 5 days. A second approach was done bytransferring positively isolated CD11c positive cells from 8-day-oldmice treated at 1 day of life with MVA-BN to naïve age matched mice. Asuspension of 2×10⁶ splenocytes containing more than 80% CD11c positivecells from MVA-BN treated mice protected naïve mice from HSV-1infection. In contrast, 4 untreated littermates, as well as 8 additionaluntreated animals, died after the challenge. Furthermore, mice receivingthe same amount of spleen cells or mice receiving one spleen equivalent(50×10⁶ cells) from the negative fraction did not show increasedresistance against HSV-1. Thus CD11c positive cells are able to protectmice from HSV-1.

After administration of MVA, short-term protective effects in the rangeof about 24 hours were described in the prior art (Vilsmeier, B., Berl.Munch. Tierärztl. Wschr. 112 (1999), 329-333). Although the viruses usedin said publication are not viruses that are not capable of beingreplicated to infectious progeny virus in the neonatal or prenatalanimal used, it was tested whether the mode of action as disclosed inVilsmeier is similar to the mode of action described in the presentapplication. More particularly, Vilsmeier discloses that MVA, inparticular inactivated MVA, induces a paramunity for about 24 hours. Totest whether the paramunity effect counts also for the protectiveeffects as disclosed in the present application mice 24 hours of birthwere vaccinated either with MVA-BN or with inactivated MVA-BN. At 7 daysof age the mice were challenged with a lethal dose of HSV-1 (10⁵ PFUHSV-1). Unvaccinated control mice died 6 days after challenge. Also themice vaccinated with inactivated MVA-BN were not protected against achallenge with HSV-1. The number of DC cells in these mice was notelevated. In contrast, the mice vaccinated with non-inactivated MVA-BNwere significantly protected against a challenge with HSV-1. 30 daysafter the challenge more than 80% of the mice were still alive. Two daysafter vaccination elevated serum Flt3-L was found in the serum. Elevatednumbers of DC were found in the spleen. The enhanced Flt3-L wasassociated with elevated numbers of DC. This confirms that paramunityeffects are not responsible for the observed protection.

(ii) MVA-BN Induces a Specific Immunity in Neonates that Lasts UntilAdulthood

One-day-old C57Bl/6 mice (group size of 18) were vaccinated (i.p) withMVA-BN (2.5×10⁷ TCID₅₀). Four weeks after vaccination, when the micewere considered adults there where challenged with a lethal dose (1×10⁴TCID₅₀) of vaccinia Western Reserve (VV-WR). With the exception of oneanimal all other MVA-BN vaccinated animals survived. In contrast, allplacebo vaccinated animals died within 7 days and demonstrated severeclinical symptoms such as ruffled fur, weight loss and reduced activity.Clearly this is a clear demonstration that MVA-BN vaccination is notonly safe in neonatal animals, but is capable of inducing a protectiveimmune response against a lethal vaccinia (related virus to MVA-BN)infection.

Example 3 (i) The Long-Term, but not Short-Term, Anti Viral Effect ofIFNα/β Depends on FL and pDC 3.1 Introduction

Treatment of newborn mice with MVA-BN increases resistance againstinfection with heterologous Herpes simplex virus type 1 (HSV-1) one weekafter MVA-BN treatment (see above). The protection is associated withincreased levels of FL in serum and increased numbers of pDC.

In this example, the role of IFNα and FL (Flt-3L) in the defense againstHSV-1 in neonatal mice was investigated. The data show that there is aFL-independent, short-term and a FL-dependent, long-term effect of IFNα.During the long-term effect, IFNα induces FL which is able to increasethe number of pDC. Although protection against HSV-1 is not solelydependent on pDC, they play an important role in the defense againstHSV-1 in neonatal mice.

3.2 Results

3.2.1 The Short-Term Protection Induced by rIFNα is FL-Independent

As previously shown (Vollstedt, 2003, J. Exp. Med. 197:575), rIFNαtreatment of C57BL/6 mice at day 6 of age, increased resistance againstHSV-1 challenge at day 7. The LD₅₀ of untreated, neonatal C57BL/6 miceis 10³ pfu of HSV-1. This LD₅₀ was increased 10-fold to 104 pfu of HSV-1after rIFNα treatment.

To analyze a possible cooperation between IFNα and FL in the defenseagainst HSV-1, FL-gene deleted (FL−/−) mice were used. It was shown thatthese animals were very susceptible to HSV-1 infection. As few as 50 pfuof HSV-1 killed 100% of 7-day-old neonates. Treatment with rIFNα at day6 of age increased resistance, so that 80% of the FL−/− mice survived5×10³ pfu HSV-1 challenge at day 7, while all untreated FL−/− neonatesdied. Thus, rIFNα is demonstrated to confer protection during aninfection with HSV-1, even in the absence of FL.

3.2.2 The Short-Term Protection Induced by MVA-BN is FL-Independent

As previously shown, MVA-BN, an efficient inducer of IFNα,β (Buttner,1995, Vet. Immunol. Immunopathol. 46:237; Vilsmeier, 1999, Berl. Munch.Tierarztl. Wochenschr. 112:329; Franchini, 2004, J. Immunol. 172:6304),is able to increase resistance to neonatal infection with HSV-1 in aIFN-dependent manner. MVA also induces FL in vivo in neonatal mice(Franchini, 2004; J. Immunol. 172:6304) Therefore, it was analyzedwhether treatment with MVA at day 5 of age also induced protectionagainst HSV-1 in FL−/− neonates. Indeed, in a dose-dependent manner,MVA-BN was able to protect against HSV-1. A dose of 2.5×10⁶ MVA-BNprotected 80% of the neonatal mice, while a dose of 2.5×10⁴ MVA-BN didnot have any protective effect.

Thus, it was concluded that rIFNα or MVA-BN have a short-term,FL-independent effect on the resistance against HSV-1 in neonatal mice.

3.2.3 The Long-Term Protection of rIFNα is FL-Dependent

MVA-BN treatment within 24 hours of birth was demonstrated to protectagainst infection with HSV-1 at day 7, when the IFNα,β system wasintact, and was demonstrated to elevate FL concentration in serum and DCnumbers in spleen in neonatal mice (Franchini, 2004, J. Immunol.172:6304). Since induction of IFNα occurred very early and viralchallenge was one week later, MVA-BN induced long-term protectionagainst viral infections was considered dependent on MVA-induced IFNαand FL. To evaluate the relative contribution of IFN and FL to thislong-term effect, treatment with rIFNα or MVA-BN at birth was evaluatedfor protection against an HSV-1 infection one week post-treatment inFL−/− mice.

C57BL/6 mice were treated with rIFNα at day 0/1 and challenged withHSV-1 at day 7. rIFNα treated mice showed an increased resistance withan LD₅₀ of 10⁵ pfu of HSV-1. To determine the dependence on FL, FL−/−neonates were treated with rIFNα at day 0/1 and then infected with 5×10³pfu HSV-1 at day 7. rIFNα treatment did not show any effect and alltreated mice died within the same time interval as untreated controls.MVA-BN treatment of FL−/− mice within 24 hours of birth also did notshow any protective effect with any dose used.

Thus, it was determined that rIFNα has a long-term, FL-dependent effecton the resistance against HSV-1 in neonatal mice.

MVA-BN not only induces IFNα, but a plethora of other cytokines, such asIL-2, IL-6, IL-12 and TNF (Buttner, 1995, Vilsmeier, 1999, see above).The production of these cytokines in the absence of FL was notdemonstrated to confer any protection against HSV-1.

3.2.4 The Absence of FL does not Impair IFNα,β Production In Vitro

The high susceptibility of FL−/− neonates to HSV-1 could have beencaused by a FL-dependent inability for IFNα,β production. The capacityof spleen cells from 7-day-old mice to produce IFNα,β in vitro wasinvestigated. After overnight cultivation with HSV-1, at an MOI of 10,spleen cells of FL−/− neonates produced 220 U/ml IFNβ, which was morethan cells from C57BL/6 neonates. Production of IFNα by spleen cells ofFL−/− neonates was 30 U/ml, while IFNα was not detected in supernatantsof stimulated cells from C57BL/6 neonates.

3.2.5 The Absence of FL Leads to Decreased Numbers of DC

To define which cell populations were responsible for the highsusceptibility of FL−/− neonates, an extensive phenotyping of spleencell populations of FL−/− neonates at day 7 was performed. DC's werehighly reduced in cell number and had a rather immature appearance withlow expression of CD11c and MHCII. No difference in NK and B cellnumbers was detected. Treatment with human (hu) or murine (mu) FLreconstituted DC to similar numbers as seen in C57BL/6 mice andincreased the relative number of NK cells.

To confirm these data, immunohistology of spleen and liver from7-day-old FL−/− and C57BL/6 neonates was performed. Numbers ofCD11c-positive cells were reduced in spleen and liver of FL−/− neonateswhen compared to C57BL/neonates.

Next, it was determined whether treatment with human and murine FL alsoreconstituted resistance in FL−/− neonates to C57BL/6 levels. Aftertreatment with huFL for a week FL−/− neonates had an increased LD₅₀ of10⁴ pfu of HSV-1, while treatment with muFL resulted in an LD₅₀ of 10³pfu of HSV-1, which equals the resistance of C57BL/6 neonatal mice.

Thus, the cells from FL-dependent progenitors, such as DC, appear toplay an important role in the defense against HSV-1 in neonatal mice.

3.2.6 rIFNα Treatment of Wild Type Neonates Increases the Number of pDC

rIFNα treatment increases the number and promotes the maturation of DC.C57BL/6 and FL−/− mice were treated with rIFNα and the relative andabsolute cell numbers of DC subpopulations in the spleen wereinvestigated.

Flow cytometry analysis revealed that rIFNα treatment of C57BL/6neonates at day 0/1 increased the number of pDC at 7 days of age. Theproportion of pDC to cDC was 0.3:1 in untreated animals, while rIFNαtreatment at this time point changed this proportion to 1.2:1. Inabsolute cell numbers, pDC numbers increased 2-fold and cDC numbersdecreased 2-fold in the spleen of day 0/1 treated neonates. Treatmentwith rIFNα at day 6 changed the proportion of pDC to cDC to 1:1.Expression of CD8α, CD4 and MHC class II was unchanged after any rIFNαtreatment.

Thus, it may be concluded that rIFNα treatment leads to an increase ofpDC numbers, which is FL-dependent.

3.2.7 rIFNα Induces Increased FL Production

Since treatment with rIFNα increased the numbers of pDC in C57BL/6neonates with an intact FL system, it was determined whether IFNα itselfcould induce FL production in these mice. Untreated 1 to 4-day-old micehad moderate levels of FL in the blood serum (450 pg/ml). rIFNαtreatment at day 1 resulted in an increase in these levels. As early as6 hours after treatment, the serum levels were 600 pg/ml, whichincreased to 1000 pg/ml after 12 hours and peaked at 1400 pg/ml at 24hours. The FL serum levels started to decrease at 48 hrs to 1300 pg/mland were close to control levels after 96 hours (700 pg).

Thus, rIFNα is able to induce a higher production of FL in the bloodserum of neonatal C57BL/6 mice.

3.2.8 Induction of pDC by FL is IFNα,β-Independent

Since rIFNα was demonstrated to increase the number of pDC, theIFNα,β-dependent development of pDC was investigated. This effect wasevaluated in IFNα,β receptor gene-deleted (A129) neonatal mice. By flowcytometry, it was demonstrated that these mice had numbers of splenicpDC similar to C57BL/6 neonates. Treatment with human or murine FLincreased the number of pDC, as well as the number of cDC similar tothat in C57BL/6 neonates (Vollstedt, 2003, see above).

Thus, although rIFNα induces increased numbers of pDC, the developmentof pDC can be augmented by exogenous FL treatment in A129 mice.

3.2.9 pDC Play an Important Role in the Defense Against HSV-1 inNeonates

IFNα and pDC have been demonstrated/observed to play an important rolein the defense against HSV-1 in neonatal mice. To test the effect of pDCdirectly, pDC from FL-treated adult mice were transferred into 6-day-oldFL−/− mice and infected with HSV-1 at day 7. Control mice usually diedaround day 5 and 6. Transfer of 5 million pDC into 6-day-old miceincreased the rate of survival so that 4 out of 10 mice survivedinfection after 3 weeks. When 5 million cDC were transferred, the onsetof death was later, within a time frame of 8 to 10 days, and only 1 outof 10 mice survived the infection after 3 weeks. Consequently, it may beconcluded that pDC are effective for the protection against lethalinfection with HSV-1.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference.

1-43. (canceled)
 44. A method for elevating the numbers of dendriticcells (DCs) in a neonate comprising increasing the level of Flt3-L inthe serum of the neonate at least 1.5 fold.
 45. The method of claim 44,wherein the neonate is a mouse.
 46. The method of claim 44, wherein theneonate is a human.
 47. The method of claim 44, wherein the level ofFlt3-L in the serum of the neonate is increased at least 2.0 fold. 48.The method of claim 44, wherein the DCs are CD11c+.
 49. The method ofclaim 44, wherein the DCs are CD45R+.
 50. The method of claim 44,wherein the DCs are CD45R+ and CD11c+.
 51. The method of claim 44,wherein the DCs are plasmocytoid dendritic cells (pDCs).
 52. The methodof claim 46, wherein the human neonate is less than 2 months old. 53.The method of claim 46, wherein the human neonate is less than 6 weeksold.
 54. The method of claim 44, wherein the Flt3-L is increased byadministering an MVA to the neonate.
 55. The method of claim 54, whereinthe MVA is MVA-BN.
 56. The method of claim 54, wherein the MVA comprisesat least one heterologous nucleic acid sequence.
 57. The method of claim56, wherein the heterologous nucleic acid sequence encodes a foreignantigen.
 58. The method of claim 56, wherein the heterologous nucleicacid sequence encodes a tumor antigen.
 59. The method of claim 55,wherein the MVA comprises at least one heterologous nucleic acidsequence.
 60. The method of claim 59, wherein the heterologous nucleicacid sequence encodes a foreign antigen.
 61. The method of claim 59,wherein the heterologous nucleic acid sequence encodes a tumor antigen.62. The method of claim 44, wherein the Flt3-L is increased byadministering Flt3-L to the neonate.
 63. The method of claim 44, whereinthe Flt3-L is increased by administering IFNα to the neonate.